: This project seeks to exploit recently developed molecular genetic tools to identify genes associated with bradyzoite differentiation in Toxoplasma gondii, a prominent opportunistic pathogen associated with AIDS. Approximately 30% of the US population is chronically infected with T. gondii, and reemergence of parasites from latent bradyzoite tissue cysts is thought to be the primary source of toxoplasmic encephalitis and other pathologies. Effective chemotherapy is available for acute infection, but immunocompromised individuals require life-long prophylaxis to guard against cyst reemergence, and chronic treatment is associated with a high frequency of adverse reactions. Treatments that target the latent bradyzoite cyst form-eliminating infection in HIV-positive individuals- would therefore be highly desirable. Previous research conducted under the auspices of this grant resulted in characterization of the bifunctional DHFR-TS enzyme of T. gondii- the primary target for chemotherapeutic management of acute toxoplasmosis- and evaluation of its role in drug resistance. Sequences derived from the DHFR-TS gene have also been employed to develop tools for molecular genetic analysis, including strategies for high frequency parasite transformation, gene cloning by complementation, production of gene "knock-outs" via targeted homologous recombination, saturation mutagenesis of the parasite genome using insertional mutagenesis vectors, and cloning of the tagged loci by plasmid rescue. These techniques have been exploited to identify several potential drug targets involved in nucleoside metabolism, including genes encoding UPRT, HXGPRT, AK, and the parasite's major adenosine transporter.Targeted deletion of the UPRT locus produces crippled parasites that permit highly efficient bradyzoite differentiation in vitro, while the HXGPRT gene has been developed as a combined positive-negative selectable marker. Using a promoter- deficient HXGPRT for insertional mutagenesis permits trapping of bradyzoite-specific promoters. This strategy will be expanded beyond the pilot studies carried out to date, to identify new genes that may identify metabolic functions to be targeted in cyst-specific chemotherapy. Bradyzoite specific genes identified by insertional mutagenesis, differential display PCR, and informatics-based approaches will be cloned and characterized, and their expression analyzed by Rnase protection, competitive RT-PCR, and as fusions with reporter genes. Additional cycles of mutagenesis will be carried out using parasites expressing HXGPRT and/or GFP selectable markers under the control of bradyzoite-specific promoters, thereby walking upstream in the genetic regulatory cascade that controls parasite differentiation. This approach should identify genes that are essential for tachyzoite differentiation into bradyzoites, cyst maintenance, and de-differentiation as bradyzoites reemerge to yield acutely infectious tachyzoites.